| Background and ObjectivesOrgan transplantation is the most effective therapy for patients with end-stage organ failure. However, acute rejection, which occurs within the first few weeks or months, remains a major cause of treatment failure. T cells, especially CD4+T cells play critical roles in the activation of the immune responses to alloantigens that cause graft rejection. Two survival pathways for T cells have been predominantly targeted: the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway and the PIM pathway. T-cell growth and survival following ligation of cytokine or antigen receptors is mediated, in part, through activation of the effector enzymes AKT and mTOR in the phosphatidylinositol3-kinase pathway and PIM kinases may provide the alternative pathway for T-cell survival. Even through the immunosuppressive therapies that block the PI3K/AK.T/mTOR pathway have been proven to be uniquely effective for anti-allotransplant in clinic, there has been no reports on the effects of Pim pathway blockade in either mice allotransplant models or human transplant recipients. This has led to the speculation that combined blockade of these two pathways might have synergistic effects in inducing immune tolerance, and thus, there has been a long-standing interest in translating about these results to the clinic. The CD4+T cells can be differentiated into various subsets. CD4+CD25+Foxp3+T cells, an important subset of CD4+T cells, can potently suppress the activation or function of conventional CD4+CD25-T effector cells. It has been reported that donor antigen-specific CD4+CD25+Foxp3+regulatory T (Treg) cells can regulate alloresponses and promote donor-specific tolerance in a skin transplantation model. Of the standard immunosuppressive agents that have been combined with Tregs, perhaps the most detailed work has been accomplished with rapamycin. Studies have shown that Tregs are preferentially able to retain their suppressive function in the presence of rapamycin. Interestingly, Foxp3induced pim2expression in natural Tregs, which caused Foxp3-expressing Treg cells to be selected in the presence of rapamycin. These studies suggested that pim2may be associated with the function of Treg cells in allograft rejection. Furthermore, in our previous study, an approximately five-fold overexpression of the pim2gene, as determined by sequencing-based serial analysis of gene expression, was detected in the allograft-activated CD4+T cells, which suggests a potential role of the Pim2kinase in allograft rejection.The pim genes represent a family of proto-oncogenes, which encode three different serine/threonine protein kinases (piml, pim2and pim3) that are related to the calcium/calmodulin kinase superfamily by weak sequence homology. pim2is an X chromosome gene (Xp11) and transcribe for three isoforms (34kD,37kD, and40kD). Pim kinases lack a regulatory domain and as a result they do not require phosphorylation for activation, and are likely to be constitutively activated following translation. The Pim kinases are regulated by rates of transcription, translation and proteosomal degradation and the STAT proteins serve as transcription factors for the pim genes following engagement of their cognate ligands. The kinase activity of these proteins is proportional to and dependent on mRNA stability, which also serves to inhibit JAK/STAT signaling through binding to the SOCS proteins, which inhibit JAK-dependent signaling. The Pim kinases play essential roles in the regulation of signal transduction cascades, included cell cycle progression, transcription factors, apoptosis and cellular metabolism and protein translation in a large variety of tissues. The pim oncogenes are over-expressed in several hematopoietic malignancies and solid cancers, and promote the cell growth, survival and drug resistance. Pim kinases showed a unique crystal structure and several specific small-molecule inhibitors currently in development are showing interesting preclinical activity in multiple cancer histology. Although pim2pathway can contribute to hematological and solid tumors developing, the physiologic role of this kinase and the precise mechanism by which inhibition of Pim2signals attenuates donor-reactive T-cell response remains uncertain. Considering of the relationship between Pim2and Treg, targeting Pim2may serve as an alternative way to induce allograft tolerance in preclinical models. Moreover, with the continuing development of Pim2-specific small-molecule inhibitors for clinical translation, immunosuppressive therapies that block the Pim2pathway would have proven to be uniquely effective in preclinical allotransplant models.Considering all of the aforementioned results, we hypothesize that Pim2kinase may participate in allograft rejection through targeting the apoptosis of CD4+T cells and modulating Treg-suppressing activities. In this study, we aimed to investigate the role and the underlying mechanism of Pim2during allograft rejection in murine skin allograft models so as to provide a new target for the diagnosis and therapy of allorejection for clinic.Part One The expression of Pim2during allograft acute rejection in murine skin allograft modelsMethods1. Establishment of mice skin graft modelsIn this model, the dorsal skin of C57BL/6mice was allotransplanted onto the BALB/c mice under sterile conditions (allogenic transplantation) and the dorsal skin of BALB/c mice was isotransplanted onto the BALB/c mice (isogenic transplantation) as the control.2. The pim2gene expression at the peak of allograft rejection analyzed with RT-PCRAt day14post-transplantation (allografts rejected completely, while isografts remained normal), pim2mRNA of splenic CD4+T cells, spleens and skin grafts of different recipient mice was examined by RT-PCR.3. The expression of Pim2protein in situ analyzed with Immunohistology stainingThe difference in the pathology of spleens and skin grafts between allorejected and isografted mice was conducted by the histology analysis. The in situ expression of Pim2protein of spleens and skin grafts from allorejected and isografted mice were conducted by immunohistochemical staining.4. The dynamic expression of Pim2protein post allotransplantation analyzed with Western BlottingThe dynamic expression of Pim2protein in spleens and grafted skins at various times post-allotransplantation was also confirmed using Western Blotting.Results1. It was found that the expression of pim2mRNA in CD4+T cells was significantly higher both in the allografted mice and rapamycin-treated allograft mice with the RT-PCR analysis, but rapamycin treatment in vivo did not significantly change the expression of pim2in allograft mice. Our results also showed that pim2mRNA of spleens and skin grafts was significantly elevated in allograft mice compared with that from isografted mice. Moreover, in allograft mice, pim2mRNA was markedly higher in the spleens than in the skin grafts, while in isografted mice, there was no significant difference between these two tissues. These data indicated that pim2gene was over-expressed when the allorejection reached the peak, suggesting that pim2gene serves as a positively related to allorejection.2. The histology observation of spleens and skin grafts from allorejected and isografted mice showed an obvious difference in the pathology between the two groups. By immunohistochemical staining, we found that Pim2protein was markedly expressed in the cytoplasm of allografted skins and spleens, but only weakly or negatively expressed in isografted skins and spleens, suggesting that Pim2was responsible for the immune-mediated tissue destruction processes.3. The immunoblotting results showed that Pim2protein was upregulated at day7in the spleens of recipient mice and reached the highest levels at day10. The accumulation of Pim2in allograft skins showed the same tendency as in the spleens, except that upregulation occurred at day10and that the highest level observed was at day14. This pattern of Pim2expression overlapped with the progression of graft rejection in response to the allogeneic antigen, indicating that Pim2kinase involved in the alloresponse and allograft rejection. Part Two The role and mechanism of Pim2in allograft acute rejectionAs shown in part one, pim2gene and protein were overexpressed during allograft rejection. Then, we further investigate how pim2work during the allorejection.Methods1. The determination of a suitable concentration and time-points for4a The spleen cells of naive B ALB/c mice were treated with different concentration of4a for different time, followed by pim2mRNA analyzed with RT-PCR2. The effect of inhibiting Pim2kinase on allograft survivalWe established the SCID mouse skin acute allograft rejection model by allografting the skin of B6mice to the SCID mice. After the wounds healed3weeks later, the alloimmune response was then reconstituted with the adoptive transfer of naive BABL/c splenic T cells treated with4a for24h or with such cells treated with DMSO as a control. The grafts were observed daily after the removal of the bandages until that the graft in control group was completely rejected.3. The potential effect of Pim2inhibiting on the apoptosis of alloreactive T cellsT cells were purified with Mouse T cell Enrichment Columns from recipient spleens at the height of allorejection, co-cultured with4a for24h and then were subjected to apoptosis analysis by flow cytometry. In addition, the phosphorylation of Bad (Ser112), a Pim2kinase substrate and apoptotic regulator, was detected by western blot in T cells treated by4a.4. The gene expression of Pim2in different CD4+T-cell subsetsBased on the finding that pim2was overexpressed in unfractionated CD4+T cells during acute allograft rejection, we analyzed the gene expression of Pim2in different CD4+T-cell subsets. After that CD4+CD25-T cells and CD4+CD25+T cells were purified and collected with MagCellect Mouse CD4+CD25+Regulatory T Cell Isolation Kit, from recipient spleens at the height of allorejection, pim2expression was detected by RT-PCR separately. Results1. Firstly, we performed RT-PCR and found that pim2mRNA was decreased in a dose-dependent manner, with a significant effect shown in the group treated with5μM4a but that little change was observed among the different treatment time points. So5μM4a and24h were chosen as the suitable concentration and time for4a.2. The T cells adoptive transfer assay showed that all of the mice in the control group (T cells treated by5μM DMSO for24h) had severe rejection at day19.5±1.7post cell transfer, while the allograft rejection in the experimental group (T cells treated by5μM4a for24h) was observed at day31±2.3. At same time, we found that there was a two-fold decrease in spleen weight compared with the control group. Therefore, blockade of Pim2significantly postponed acute rejection mediated by T cells.3. The result demonstrated a notable increase in apoptotic cells in the4a-treated group. In addition, the phosphoryla-tion of Bad (Ser112), a Pim2kinase substrate and apoptotic regulator, was significantly reduced in the4a-treated group. These results suggested that the absence of Pim2activity increased the level of T-cell apoptosis by reducing the phosphorylation of apoptotic regulator Bad (Ser112) during allograft rejection, which indicates that pim2may promote the allograft rejection by inhibiting T-cell apoptosis.4. By RT-PCR analysis of Pim2expression in different CD4+T-cell subsets during acute allograft rejection, we found that expression of pim2mRNA in CD4+CD25-T cells was2.66-fold higher than that in CD4+CD25+T cells, indicating that Pim2kinase more strongly promoted CD4+CD25-T-cell survival.Part Three The effect of Pim2on the alloantigen-induced CD4+CD25+T cellsConsidering the negative regulation of CD4+CD25+T cells observed during allorejection and the expression of pim2gene in alloantigen-induced CD4+CD25+T cells as shown in part two, we further investigated the effect of Pim2on this subset. Methods1. The effect of Pim2blocking on Foxp3gene expression analyzed with RT-PCR Gene expression of Foxp3in splenic T cells isolated from the allograft mice and treated with different concentration of4a for24h was analyzed by RT-PCR.2. The effect of Pim2blocking on the function of alloantigen-induced CD4+CD25+T cells analyzed by Flow cytometryNext, we explored whether Pim2was required for alloantigen-induced CD4+CD25+T-cell suppression activity. CD4+CD25-T cells and CD4+CD25+T cells were purified from the freshly isolated spleens of allograft mice with rejection. CD4+CD25+T cells treated with or without4a for24h were cocultured with CD4+CD25-T cells for12h at a ratio of1:6separately, and then examined for cell apoptosis by flow cytometric analysis.3. The effect of Pim2blocking on the preferential expansion of alloantigen-induced Tregs in the presence of rapamycin analyzed by Flow cytometrySince rapamycin selectively allowed for proliferation and fostering the suppressive function of Foxp3+Treg cells, next we explored whether Pim2kinase contributed to the observed an enrichment of circulating CD4+CD25+Foxp3+Treg in the presence of rapamycin. Flow cytometry of the surface expression of CD4, CD25and Foxp3on spleen cells from rapamycin-treated allografted mice was conducted after stimulated for12h with anti-pim2or with4a.Results1. By analyzing the expression of Foxp3and pim2in splenic T cells isolated from the allograft mice and treated with5or10μM4a for24h by RT-PCR, we found that4a treatment resulted in the significant decrease of Foxp3and pim2expression. Since Foxp3is the key transcriptional factor of CD4+CD25+T cells, the result showed that blockage of pim2by4a reduced the proliferation of alloantigen induced CD4+CD25+T cells, indicating that Pim2is indispensable for the ex vivo activation of alloantigen-induced CD4+CD25+T cells.2. The results of flow cytometric analysis showed that the alloantigen induced CD4+CD25+T cells treated with5μM4a led to an obvious decrease in the percentage of alloantigen induced CD4+CD25-T-cell apoptosis compared with those untreated with4a, suggesting that the blocking of Pim2impaired the function of CD4+CD25+T cells to induce CD4+CD25-T-cell apoptosis. The data indicated that Pim2is indispensable for the ex vivo function of alloantigen-induced CD4+CD25+T cells.3. The results of flow cytometric analysis showed that treatment with either anti-pim2or4a led to a substantially reduced expansion of the observed enrichment of CD4+CD25+Foxp3+Treg in the presence of rapamycin alone or combined with cycloheximide, suggesting that pim2allowed alloantigen-induced CD4+CD25+T cells to evade many rapamycin-imposed signaling blocks and to expand preferentially.Part Four Cycloheximide served as an immunosuppressive drug to prevent allorejection through the pim2pathwayConsidering that cycloheximide could inhibit pim2to function in anti-apoptosis, and the blockade of Pim2prolonged skin graft survival through the apoptosis regulation pathway in our previous study, we hypothesize that cycloheximide may prolong skin graft survival.’Methods1. The effect of cycloheximide in inhibiting pim2in vitro analyzed by RT-PCR For determination of a suitable concentration of cycloheximide, spleen T cells from naive BALB/c mice were treated with different concentrations of cycloheximide for24h, followed by RT-PCR analysis of pim2mRNA.2. The effect of blocking pim2by cycloheximide on the allograft survival We established the SCID mouse skin acute allograft rejection model by allografting the skin of B6mice to the SCID mice. After the wounds healed3weeks later, the alloimmune response was then reconstituted with the adoptive transfer of naive BABL/c splenic T cells treated with cycloheximide for24h or with such cells treated with DMSO as a control. The grafts were observed daily after the removal of the bandages until that the graft in control group was completely rejected.3. The effect of cycloheximide administrated in vivo on the allograft survivalWe also confirmed the above result by in vivo administration of cycloheximide daily post allotransplantation. After the establishment of BALB/c mice skin allograft models, we performed the administration of cycloheximide, rapamycin and the combination of cycloheximide and rapamycin via intraperitoneal injection and then observed the grafts daily.4. Flow cytometry analysis of the effect of cycloheximide combined with rapamycinBy the use of flow cytometry, we further investigated the immunological effect on alloantigen-reactive CD4+T-cells expansion of the joint administration of cycloheximide and rapamycin. To understand whether cycloheximide resulted in the expansion of Treg, we used the flow cytometry to analyze the frequency of Foxp3Treg cells in the spleens of cycloheximide-treated mice when the control mice developed a donor reactive T-cell response peaked.Results1. We found that Pim2mRNA was decreased by cycloheximide in a dose-dependent manner, with a significant effect shown in the group treated with50μM cycloheximide for24h.2. The result of T cells adoptive transfer assay showed that all of the allograft SCID mice in the control group (T cells treated by50μM DMSO for24h) had severe rejection at day19.0±1.00post cell transfer, while the allograft rejection in the experimental group (T cells treated by50μM cycloheximide for24h) was observed at day26.3±1.53. The result suggested that cycloheximide significantly postponed acute rejection mediated by T cells through blockade of Pim2.The in vivo administration of cycloheximide also showed that in the allogeneic BABL/c models, control mice rapidly rejected allograft with a median survival time (MST) of12.4±1.10d, whereas treatment with either cycloheximide or rapamycin significantly prolonged survival to more than17.6±0.89d and19.6±1.52d respectively. However, although the combination of cycloheximide and rapamycin has been shown to significantly delay rejection (16.8±1.64), it was no more effective in inducing allograft tolerance than cycloheximide alone. The survival advantage conferred by treatment with cycloheximide provides further evidence for the importance of cycloheximide in preventing alloimmune responses.3.The results of flow cytometry analysis of the spleen of transplanted and treated mice revealed that the number and the frequency of circulating alloantigen-reactive CD4+T cells was reduced both in mice treated with the combined protocol (from8%to3.2%)and in those treated with rapamycin alone (from8%to2.8%). Nevertheless, treatment with cycloheximide alone in vivo did not led to a substantially reduced accumulation of donor-reactive CD4+T-cells compared with untreated controls(p>0.05), suggesting there may exist another mechanism for cycloheximide to postpone allorejection. Notably, no differences in the total number of CD4+T cells were observed among untreated and treated mice in vitro by either anti-Pim2or4a. Finally, the flow cytometry analyses showed an obvious increase of Foxp3+Treg cells in the spleen of mice treated both with the combined protocol (from4.3%to7.6%) and with rapamycin alone (from4.3%to5.6%). In contrast, we found slight decrease in the frequency of CD4+CD25+Foxp3+Treg cells in mice treated with cycloheximide alone (from4.3%to3.2%) compared with untreated controls. Interestingly, the combination of cycloheximide/rapamycin treatment in vivo led to a dramatic increase in the frequency of CD4+CD25+Foxp3+Treg compared with treatment by rapamycin alone (p<0.05) which has opened a new method for the ex vivo expansion of CD4+CD25+Foxp3+Treg.Conclusions1. Pim2was highly expressed in allografted mice at the peak of rejection and the pattern of Pim2expression overlapped with the progression of graft rejection in response to the allogeneic antigen, suggesting that Pim2played important roles in the alloresponse and graft survival and mediated tissue destruction processes during allograft rejection. 2. Blockade of Pim2kinase by4a significantly prolonged the allograft survival through reducing the phosphorylation of apoptotic regulator Bad (Ser112) so as to induce the apoptosis of the alloreactive T cells, indicating that pim2may promote the allograft rejection by inhibiting T-cell apoptosis.3. Pim2enforced an alloantigen-induced CD4+CD25+Foxp3+T-cell phenotype and promoted the ex vivo expansion of CD4+CD25+Foxp3+T cells. Alloantigen-induced CD4+CD25+T cells appear to display high suppressive activity in the presence of Pim2. Pim2blockade inhibits alloantigen-reactive CD4+CD25+Foxp3+Treg expansion in the presence of rapamycin, indicating that the pim2pathway was partly responsible for the expansion of CD4+CD25+Foxp3+Treg observed in the presence of rapamycin. Collectively, Pim2is indispensable for the ex vivo activation and function of alloantigen-induced CD4+CD25+T cells, which has opened a new method for the ex vivo expansion of CD4+CD25+T cells.4. Cycloheximide was able to prolong skin allograft survival through inhibition of pim2, with little effect on the expansion of CD4+T cells or CD4+CD25+Foxp3+Treg. However cycloheximide could significantly enhance the ability of rapamycin to expanse CD4+CD25+Foxp3+Treg. Although treatment with the combined protocol led to increased frequency of splenic CD4+CD25+Foxp3+Treg, it was no more efficient in controlling graft rejection than the alone protocol.Innovations1. It is the first time to investigate the role of pim2in murine skin allograft models. We confirmed that Pim2gene and protein were highly expressed in allografted mice and pim2may promote the allograft rejection by inhibiting T-cell apoptosis in murine skin allograft models. These results provide a mean to further understand the mechanisms and developing therapeutic approaches to prevent allograft acute rejection. 2. It is the first time to characterize the relationship between pim2kinase and CD4+CD25+Foxp3+Treg expansion. We confirmed that Pim2enforced an alloantigen-induced CD4+CD25+Foxp3+T-cell phenotype and Treg-mediated suppression may be partly related to pim2pathway. Pim2pathway was partly responsible for the expansion of CD4+CD25+Foxp3+Treg observed in the presence of rapamycin. Moreover, cycloheximide could significantly enhance the ability of rapamycin to expanse CD4+CD25+Foxp3+Treg. These results provide a mean to optimize the methods for ex vivo expansion of CD4+CD25+Foxp3+Treg cells and promote Foxp3+Treg-based immunosuppression strategies.3. The allograft survival was successfully prolonged by the use of4a or Cycloheximide and we found that both of the two drugs prevented the allograft rejection partly through blockade of Pim2kinase. The survival advantage performed by treatment with4a or cycloheximide provides further evidence for the importance of blockade of the pim2pathway in preventing alloimmune responses. |
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